Dihydrofolate reductase (DHFR) is the target enzyme for the widely used cancer chemotherapeutic drug methotrexate (MTX). We are studying the molecular mechanism for regulating DHFR gene expression in mammalian cells. Previous studies in my laboratory have shown that the expression of this gene is regulated in a well-defined manner and over a wide range when resting cells are induced to re-enter the cell cycle. We have shown that DHFR gene expression is regulated in a manner which is quite different from that of any other gene which has been studied in detail so far. In this proposal we describe experiments which will determine directly if the cell regulates DHFR gene expression by controlling the amount and/or translation of DHFR mRNA. We will also determine if the export of DHFR mRNA from the nucleus to the cytoplasm is regulated. DHFR mRNA will be quantitated either by determining the ability of a mRNA preparation to direct the synthesis of DHFR in an in vitro mRNA-dependent translating system or by analyzing the kinetics of hybridization of DHFR cDNA to an RNA preparation. These studies will be conducted with a MTX-resistant 3T6 cell line (M50L3) which we recently isolated. The cell line overproduces DHFR and its mRNA (and presumably the DHFR gene as well) by a factor of at least 200 compared to normal 3T6 cells. The use of this cell line will greatly facilitate the isolation and quantitation of both the enzyme and its mRNA. We have found that DHFR gene expression appears to be regulated in the same manner in the overproducing cells as in normal 3T6 cells. Our results will lead to a better understanding of the molecular basis for the differential toxic effects of MTX toward normal vs rapidly growing or tumor cells.